UC-NRLF 


B    3    1E7    TD7 


EXCHANGE 


1.9  '974 


A  Critical  Investigation  and  an  Application 

of  the  Rat  Growth  Method  for  the 

Study  of  Vitamin  B. 


BY 

ADELAIDE  SPOHN 


Submitted  in  Partial  Fulfillment  of  the  Requirements 

for  the  Degree  of  Doctor  of  Philosophy  in 

the  Faculty  of  Pure  Science, 

Columbia  University. 


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(O 

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NEW  YORK 
1922 


A  Critical  Investigation  and  an  Application 

of  the  Rat  Growth  Method  for  the 

Study  of  Vitamin  B. 


BY 

ADELAIDE  SPOHN 


Submitted  in  Partial  Fulfillment  of  the  Requirements 

for  the  Degree  of  Doctor  of  Philosophy  in 

the  Faculty  of  Pure  Science, 

Columbia  University. 


NEW  YORK 
1922 


ACKNOWLEDGMENTS 


This  investigation  was  undertaken  at  the  sugges- 
tion of  Professor  H.  C.  Sherman  and  carried  out 
under  his  direction.  The  author  wishes  to  express 
to  Professor  Sherman  her  appreciation  for  advice 
and  encouragement  received  during  the  course  of  the 
work. 

The  author  wishes  also  to  express  thanks  to  Pro- 
fessor A.  W.  Thomas  for  helpful  suggestions. 

BXGNANGK 


CONTENTS 


I.     Introduction. 

II.     Experimental  Procedure. 

1.  Selection  and  Care  of  Animals. 

2.  Basal  Ration  and  its  Preparation. 

3.  Source   of   Vitamin   B,   Treatment    and   Manner   in 

Which  it  Was  Fed. 

III.  The  Adequacy  of  the  Basal  Diet. 

IV.  Determination    of    the    most    Advantageous    Dose    for 

Measuring  Heat  Destruction  of  Vitamin  B  in  Skim- 
med Milk  Powder. 

1.  Feeding  the  Milk  Separately  from  the  Rest  of  the 

Diet. 

2.  Feeding  the  Dried  Milk  Mixed  with  the  Rest  of  the 

Diet. 

V.     The  Effect  of  Heat  upon  Vitamin  B  in  Milk  in  the  Dry 
and  Fluid  State. 

1.  Experiments  with  Reconstructed  Milk  Fed  Separate- 
ly from  the  Rest  of  the  Diet. 

2.  Experiments  with  Milk  Mixed  with  the  Rest  of  the 
Diet. 

VI.     Summary. 


A  CRITICAL  INVESTIGATION  AND  AN  APPLICATION 

OF  THE  RAT  GROWTH  METHOD  FOR  THE 

STUDY  OF  VITAMIN  B. 


INTRODUCTION 

The  term  "vitamin  B"  is  here  used  synonomously  with 
"water  soluble  B"  as  a  designation  for  the  water  soluble 
growth  promoting  substance  whose  existence  as  an  essential 
constituent  of  the  food  was  demonstrated  by  Hopkins  x  &  2, 
Osborne  and  Mendel 3'7,  and  McCollum  and  Davis 8'11  by 
growth  experiments  with  young  mammals,  chiefly  rats. 
Whether  this  is  identical  with  the  antineuritic  substance  stud- 
ied by  Eijkman 12-14,  Funk15-16,  Williams17'20  and  others 
through  experiments  with  birds,  or  with  the  "bios"  of  Wil- 
diers  21  which  shows  a  growth  promoting  property  toward 
yeast,  does  not  concern  the  present  investigation.  The  recent 
publication  of  a  monograph  The  Vitamins  by  the  American 
Chemical  Society  22  makes  it  unnecessary  to  review  previous 
work  at  length  in  this  connection. 

The  importance  of  vitamin  B  as  a  factor  in  food  values 
makes  desirable  the  standardization  of  the  method  of  studying 
this  substance  so  as  to  permit  of  quantitative  investigation  of 
the  relative  amounts  in  different  foods  or  in  the  same  food 
before  and  after  heating  or  other  treatment  to  which  foods 
may  be  subjected  in  the  course  of  preservation  or  cooking. 

When  such  methods  are  sufficiently  developed  they  can 
also  be  applied  to  the  quantitative  measurement  of  the  concen- 
tration of  the  vitamin  at  successive  steps  in  attempts  to  iso- 
late the  substance  from  natural  sources  and  bring  it  to  a  con- 
dition of  maximum  purity  for  the  determination  of  its  chemi-. 
cal  nature. 

For  the  present  investigation  white  rats  were  selected  as 
the  experimental  animal  and  dried  skimmed  milk  as  the  vita- 
min-containing food.  Examination  of  the  literature,  as  well 


as  the  author's  experience,  shows  that  the  pigeon  method  for 
studying1  the  vitamin  B  content  of  food,  by  the  power  of  the 
substance  to  prevent  or  cure  polyneuritis,  yields  rather  irregu- 
lar results15'  22>  36>  38.  The  method  of  testing  for  vitamin  B 
by  measuring  the  acceleration  of  yeast  growth  is  open  to  the 
question  whether  the  culture  medium  is  adequate  in  all  other 
respects  and  whether  it  is  safe  to  assume  that  increased  yeast 
is  attributable  entirely  to  the  vitamin  23-35.  The  nutrition  re- 
quirements and  rate  of  growth  of  'the  rat  have  been  exten- 
sively studied  by  Hopkins  *»  2j  36,  Osborne  and  Mendel  3~7, 
McCollum  8-11  and  others.  The  rat  method  is  therefore  the 
best  standardized  by  previous  work.  Dr.  Harriet  Edge- 
worth  37,  working  in  this  laboratory,  after  a  careful  analysis 
of  the  yeast  and  rat  growth  methods  concluded :  "The  rat 
growth  method  involves  somewhat  larger  probable  errors  than 
the  yeast  method,  but  can  be  interpreted  in  terms  of  B  vitamin 
with  much  greater  certainty  and  is  therefore  the  preferable 
method."  Skimmed  milk  in  the  form  of  dry  powder  was 
chosen  as  the  source  of  vitamin  B,  since  it  furnishes  the  vita- 
min in  a  typical  natural  state,  readily  available  and  uniform. 
It  is  easily  and  accurately  manipulated  in  either  large  or  small 
quantities  and  is  convenient  for  study  in  either  the  fluid  or 
dry  state. 


EXPERIMENTAL  PROCEDURE 

Selection  and  Care  of  Animals.  The  rats  used  were  twenty- 
eight  or  twenty-nine  days  old,  this  being  the  time  of  weaning 
adopted  in  this  laboratory,  and  weighed  from  thirty  to  sixty- 
five  grams  when  placed  on  the  experimental  diet.  The  experi- 
mental animals  all  came  from  mothers  on  a  diet  of  two-thirds 
whole  wheat,  one-third  whole  milk  and  sodium  chloride,  two 
per  cent,  of  the  weight  of  the  wheat  (diet  13)  or  this  diet  with 
an  addition  of  ten  grams  of  raw  lean  beef  per  adult  rat  per 
day  (diet  13M).  In  each  experiment,  however,  care  was 
taken,  that  equal  numbers  of  comparison  and  control  rats 
came  from  mothers  on  the  same  diet.  The  lots  were  made  up 
in  the  following  manner.  If,  for  example,  in  one  experiment 


there  were  five  variations  of  the  diet  and  ten  rats  -for  each 
variation,  then  fifty  rats  were  required.  Six  to  eight  litters  of 
from  five  to  ten  animals  each  would  be  needed  to  supply  this 
number  of  rats.  Each  litter  was  distributed  over  the  five  vari- 
ations as  far  as  it  would  go.  For  example,  a  litter  of  nine  rats 
would  furnish  two  animals  each  for  four  variations  and  one 
for  the  fifth.  Then  a  litter  of  six  rats  would  provide  for  one 
rat  where  there  were  two  before,  and  two  rats  where  there  was 
one  before,  making  a  total  of  three  rats  for  each  variation  of 
the  diet.  The  remaining  thirty-five  rats  from  the  various  lit- 
ters were  portioned  out  so  that  there  were  ten  animals  in  each 
lot,  and  in  such  a  way  that  the  total  weight  of  the  rats  and  the 
number  of  males  and  females  on  each  modification  was  prac- 
tically the  same. 

The  experiments  were  continued  for  eight  weeks,  from 
the  begining  of  the  fifth  to  the  end  of  the  twelfth  week  of  the 
rat's  life,  this  being  the  most  rapid  growth  period.  A  number 
of  experiments  were  continued  for  four  weeks  longer.  These 
last  four  weeks,  however,  did  not  show  any  significant  differ- 
ences which  were  not  brought  out  in  the  growth  curves  from 
the  fourth  through  the  twelfth  week. 

When  the  milk,  which  was  the  source  of  vitamin  B,  was 
fed  separately  from  the  rest  of  the  diet,  the  animals  were  kept 
in  round  galvanized  iron  wire  cages,  eight  inches  high  and 
nine  inches  in  diameter,  one  rat  in  each  cage.  In  addition  to 
the  milk  these  rats  received  a  vitamin  B-free  basal  ration,  of 
which  they  ate  ad  libitum.  In  the  experiments  in  which  the 
rats  received  the  milk  mixed  with  the  rest  of  the  diet,  which 
was  always  before  them,  six  to  eight  animals  were  housed  in 
square  iron  wire  cages  11"  x  14"  x  10".  Fresh  water  was 
always  available.  The  cages  were  cleaned  as  often  as  seemed 
necessary,  the  large  cages  usually  every  day  and  the  small 
round  cages  three  times  a  week.  Each  animal  was  weighed 
once  a  week  and  the  food  remaining  weighed  and  subtracted 
from  the  amount  fed,  the  difference  being  the  amount  con- 
sumed by  the  rat  or  rats  during  that  week. 

Basal  Ration  and  its  Preparation.  The  basal  ration  used 
was  planned  to  contain  all  the  essential  food  factors  in  optimum 


amounts,  with  the  exception  of  vitamin  B  which  was  lacking. 
It  had  the  following  composition : 

Diet  94 

per  cent. 

Casein      -  18 

Butter  fat  10 

Starch  68 

Salt  mixture  (Osborne  and  Mendel)40      -          4 

Later,  merely  as  a  factor  of  safety,  cod  liver  oil  was  substi- 
tuted for  one-fifth  of 'the  butter  fat,  making  diet  107.  The  in- 
gredients of  the  diet  were  weighed  out  and  intimately  mixed 
first  by  hand  and  then  transferred  to  a  mechanical  mixer  and 
stirred  for  five  minutes. 

The  casein  was  prepared  from  the  commercial  product 
by  purification  in  the  manner  suggested  by  Sherman  and  Husa 
(unpublished  experiments)  as  follows :  One  liter  of  60  per 
cent,  alcohol  (by  weight)  was  added  to  200  grams  of  casein 
and  the  mixture  stirred  for  one-half  hour  by  means  of  a 
mechanical  stirrer  and  was  then  allowed  to  stand  for  five  and 
one-half  hours.  After  this  period  the  casein  was  filtered  off 
through  a  Buchner  funnel  and  washed  once  in  the  funnel 
with  500  cc.  of  60  per  cent,  alcohol.  The  casein  was  again 
treated  with  one  liter  of  60  per  cent,  alcohol  as  before  but  this 
time  was  left  to  stand  for  eighteen  hours.  It  was  filtered  off 
and  washed  as  before  with  500  cc.  of  60  per  cent,  alcohol  and 
finally  with  500  cc.  of  90  per  cent,  alcohol.  The  last  washing 
merely  facilitates  the  drying.  The  casein  was  removed  from 
the  filter  and  spread  out  in  a  thin  layer  and  allowed  to  become 
air-dry.  Throughout  this  paper  the  strength  of  alcohol  used 
is  always  given  in  per  cent,  by  weight. 

The  butter  was  melted  at  a  temperature  not  exceeding 
45°  C.  All  the  curd,  salt  and  water  settles  to  the  bottom  of 
the  vessel  and  may  be  easily  removed  when  the  butter  solidi- 
fies. The  butter  fat  is  again  melted  at  a  low  temperature,  and 
filtered  through  filter  paper  using  a  hot  water  funnel. 

The  salt  mixture  described  by  Osborne  and  Mendel 40 
was  used.  Commercially  pure  corn  starch,  which  according 

8 


to    these   authors    contains    no    appreciable  quantities  of  the 
water    soluble    vitamin    furnished    the    carbohydrate    of   the 

diet39. 

Source  of  Vitamin  B,  Treatment  and  Manner  in  Which  it 
Was  Fed.  Dried  skimmed  milk  was  used  as  a  convenient  source 
of  vitamin  B.  It  permits  the  study  of  the  effect  of  heat  on  the 
vitamin  both  in  the  wet  and  dry  state.  By  using  skimmed 
milk  powder  from  the  same  lot  throughout  one  experiment, 
possible  variation  in  vitamin  content  is  eliminated. 

The  dry  milk  was  heated  at  100°  C.  for  six,  twenty-four 
and  forty-eight  hours  in  a  constant  temperature  oven.  It  was 
spread  out  in  thin  layers  about  one-half  inch  in  thickness  and 
the  temperature  taken  by  means  of  a  thermometer  extending 
into  the  dry  milk.  Two  hours  were  allowed,  which  was  the 
time  found  to  be  necessary  for  the  milk  to  come  to  100°  C. 
The  portions  heated  six,  twenty-four  and  forty-eight  hours 
respectively  actually  remained  in  the  oven  eight,  twenty-six 
and  fifty  hours. 

The  reconstructed  milk  was  prepared  by  weighing  out 
100  grams  of  the  air-dry  powder  and  making  it  up  to  1,000  cc. 
with*  water.  For  the  heat  treatment  250  cc.  of  this  was  poured 
into  an  Erlenmeyer  flask  and  tightly  stoppered  with  cotton. 
This  was  heated  in  a  vigorously  boiling  water  bath  for  six 
hours.  A  thermometer  extending  into  the  milk  actually  re- 
corded a  temperature  of  99.5°  C.  to  99.8°  C.  At  the  end  of 
six  hours  the  flask  was  removed  from  the  bath  and  rapidly 
cooled  under  the  tap.  Since  the  volume  diminished,  due  to 
evaporation  through  the  stopper,  the  milk  was  again  made  up 
to  volume  before  using. 

The  milk  was  fed  in  doses  of  8  cc.  per  rat  per  day  for 
seven  days  a  week,  this,  as  will  later,  be  shown,  being  the 
amount  which  would  best  reveal  any  variation  in  vitamin  con- 
tent. The  milk  was  measured  into  a  small  cup  by  means  of  a 
graduated  pipette  and  placed  into  the  cage  each  day.  If  the 
rat  did  not  drink  the  milk  readily,  the  water  cup  was  removed 
for  the  day  and  replaced  in  the  evening.  Usually  this  treat- 
ment brought  the  desired  results.  After  two  to  three  days  all 
the  rats  took  all  the-  milk  that  was  given  them.  The  milk 


heated  dry  was  fed  in  two  ways:  (1)  mixed  with  the  rest  of 
the  diet,  replacing  25  per  cent,  of  the  starch  in  diet  94.  This 
ration  was  always  available  and  the  rats  ate  ad  libitum.  (2)  In 
another  series  of  experiments  the  milk,  which  was  heated  dry, 
was  reconstructed  before  feeding  by  making  25  grams  up  to  a 
volume  of  250  cc.  with  water.  This  was  fed  in  doses  of  8  cc. 
per  rat  per  day.  The  advantage  here  is  that  all  the  rats  re- 
ceived exactly  the  same  amount  of  vitamin  per  day.  Positive 
controls,  animals  receiving  in  the  one  case  the  diet  containing 
25  per  cent,  of  unheated  milk  and  in  the  other  case  8  cc.  per 
day  of  the  reconstructed  unheated  milk,  were  run  in  every  case 
in  comparison  with  the  rats  receiving  the  heated  food.  Nega- 
tive controls,  receiving  only  the  vitamin-free  diet  were  run  in 
almost  all  but  not  every  experiment.  This  was  done  in  order 
to  make  sure  that  the  basal  diet  did  not  vary. 

THE  ADEQUACY  OF  THE  BASAL  DIET 

In  order  to  prove  that  the  basal  ration  contained  the 
optimum  amounts  of  the  various  constituents,  it  was  fed 
to  a  series  of  rats  in  comparison  with  diets  in  which 
each  ingredient  of  the  basal  ration,  the  casein,  butter 
fat  and  salt  mixture,  were  in  turn  used  in  larger  amounts. 
It  was  thought  that  perhaps  another  protein  such  as  ex- 
tracted meat  might  prove  to  be  a  more  adequate  protein, 
or  the  meat  might  be  more  palatable  and  that  therefore  the 
rats  would  eat  more  of  the  basal  ration.  Consequently  diet  94 
was  also  compared  with  a  diet  in  which  extracted  meat  residue 
replaced  the  casein.  Commercial  extracted  meat  residue  was 
purified  according  to  the  method  described  for  casein  (page  7). 

TABLE   I. 
The  following  diets  were  used  in  the  comparison : 

Diet    94  102  103  105  104 

%  %  %  %  % 

Casein 18  18  -23  18  18* 

Butter  fat 10  15  10  10  10 

Starch    68  63  63  67  68 

vSalt  mixture  (O.  &M.)40....       44454 
*  Meat  residue. 

10 


The  average  growth  curves  on  these  various  diets  were 
practically  identical  and  the  average  duration  of  life  varied  by 
only  four  days.  Table  II  briefly  summarizes  the  results  of 
this  series  of  experiments. 

TABLE  II. 
Summary  of  average  results  on  diets  94,  102,  103,  104,  105. 

Number      Average          Average         Average 

of  initial  weight  age  at 

Diet  rats  weight  at  death  death 

94 12          47  gms.          34  gms.          61  days 

102 8          46    "  31     "  65    " 

103 8          47    "  34    "  64    " 

105 8          47    "  34    "  63    " 

104 8          46    "  33     "  62    " 

It  was  thought  that  the  results  might  be  different  if  vita- 
min B  were  added  to  the  diets,  in  other  words  if  this  vitamin 
was  not  the  limiting  factor  in  the  diet.  At  about  this  time  in 
the  course  of  the  work,  it  was  also  thought  wise,  merely  as  a 
factor  of  safety,  to  replace  2  per  cent,  of  the  butter  fat  in  the 
diet  by  cod  liver  oil.  Each  rat  in  this  comparison  received 
either  diet  107,  108,  109,  or  110  with  an  addition  of  7  cc.  or 
8  cc.  of  reconstructed  skimmed  milk.  These  doses  were  used, 
since  from  experiments  reported  later  in  the  paper  it  became 
apparent  that  one  or  the  other  of  these  amounts  would  prob- 
ably give  the  best  results  in  the  study  of  the  destruction  of 
water-soluble  B  by  heat.  8  cc.  was  later  decided  upon 
as  the  best  dose  but  the  results  for  both  are  here  given,  both 
being  valuable  in  this  connection. 

TABLE   III. 
The  following  diets  were  used  in  the  second  comparison: 

Diet   107        108        109        110 

%  %  %  % 

Casein    18  18          23  18 

Butter  fat    8  13  8  8 

Cod  liver  oil 2  2  2  2 

Starch    68          63          63          67 

Salt  mixture  (O.  &  M.)40 4445 

11 


Table  IV  gives  a  summary  of  the  results  of  the  compari- 
son of  diet  107  with  diet  108  containing  5  per  cent,  more  but- 
ter fat,  diet  109  containing  5  per  cent,  more  casein  and  diet  110 
with  an  excess  of  1  per  cent,  salt  mixture  over  that  in  diet  107. 

TABLE   IV. 


i  -0 

•S.e-8 
1313 

(rt 

.Is 

i^-s 

2^,* 

•£  <u  rz 

2 

"3 

'£J2S 

Aver,  age  at  death 

Gain  for 

C/3     .j  *^ 

g  rt  6 

*0 

'^*E 

u  §3  « 

or  when  killed 

8  weeks 

t> 

aj  "^   <u 

6 

>  'S 

^  *^  _c 

Q 

«  §  6 

<I  £ 

J?  "^    fe 

107 

0 

11 

47  gms. 

36  gms. 

54  days  at  death 

108 

0 

10 

47 

36    " 

55     "      "      " 

109 

0 

10 

47 

36    " 

55     "      "      " 

110 

0 

9 

47 

34    " 

53     "      "      " 

107 

7cc. 

9 

48 

69    " 

Killed  at  12  weeks 

21  gms. 

108 

7cc. 

10 

47 

84*  " 

1  dead  at  64  davs 

37    " 

Rest  killed  at  12  wks. 

109 

7cc. 

11 

49    « 

73    " 

Killed  at  12  weeks 

23 

110 

7cc. 

10 

46    " 

70    " 

'       "    12      " 

24 

107 

8cc. 

10 

47    " 

82    " 

'       "    12      " 

35 

108 

8cc. 

12 

48    " 

81    " 

'       "    12      " 

33 

109 

8cc. 

11 

47    " 

82    " 

«       «<    ^2      " 

35 

110 

8cc. 

11 

47    M 

78    " 

,       »   12      « 

31 

*  84  gms.  is  the  average  weight  of  9  rats  at  12  weeks  of  age. 

The  results  of  the  experiments  tabulated  in  Table  IV  are 
the  same  as  those  in  the  previous  series  (Table  II).  "  It  ap- 
pears from  the  data  that  the  basal  rations  94  and  107  furnish 
optimum  amounts  of  the  essential  food  factors,  protein,  fat, 
mineral  salts  and  vitamin  A. 


DETERMINATION  OF  THE  MOST  ADVANTAGEOUS 

DOSE   FOR  MEASURING  HEAT  DESTRUCTION 

OF  VITAMIN  B  IN  SKIMMED  MILK  POWDER. 

Feeding  the  Milk  Separately  from  the  Rest  of  the  Diet.  It 

was  next  necessary  to  determine  the  amount  of  milk  to  feed  in 
order  to  detect*  most  readily  any  change  in  the  water  soluble 
B  content  upon  heating  the  milk.  Increasing  amounts  of  milk 
were  superimposed  on  the  basal  diet  and  a  study  made  of  the 
growth  curves  in  relation  to  size  of  the  dose.  The  milk  was 

12 


fed  in  doses  of  2  cc.,  4  cc.,  6  cc.,  8  cc.,  10  cc.,  12  cc.,  15  cc.,  per 
rat  per  day.  These  amounts  were  fed  without  regard  to  the 
weight  of  the  rat  and  the  same  amount  was  fed  throughout 
the  experimental  period.  The  data  are  summarized  in  Tables 
V  and  VI  and  Chart  I,  Figures  I  and  II. 

TABLE  V. 
Diet  94  and  unheated  milk. 


No. 
of 
rats 

6 

Milk 
cc.       4 
per 
day 
2      50 

Age  in  weeks 
5      6      7     8      9     10    11 

Average  weight  in  grams 
57    61    60    57    53* 

12 

Total 
£f8         Remarks 

wks. 
see  (1)  below 

6 

4 

47 

58 

60 

56 

53 

50* 

see  (2)  below 

5 

6 

44 

52 

59 

64 

63 

63 

62    63 

66 

22 

3 

8 

45 

54 

66 

77 

86 

95 

99  108 

116 

71 

All  rats  K.  at 

6 
6 

10 
12 

45 
46 

56 
58 

67 
74 

76 
84 

85 
92 

94 
99 

99  101 
99  103 

107 
113 

62 
67 

end  of  12th  wk. 

6 

15 

48 

65 

81 

90 

100 

108 

107  111 

110 

62 

K.  =  killed.  D.  =  dead. 

*  Since  all  rats  did  not  live  till  the  end  of  the  experimental  period, 
averages  could  not  be  given  for  the  last  three  weeks. 

(1)  4  rats  D.  at  64,  65,  77,  77  days. 
2  rats  K.  at  end  of  12th  wk. 

(2)  4  rats  D.  at  63,  66,  70,  74  days. 
2  rats  K.  at  end  of  12th  wk. 


TABLE  VI. 
Diet  107  and  unheated  milk. 


No. 
of 
rats 

Milk 
cc. 
per 
day 

4 

Age  in  weeks 
5   6   7   8   9   10  11 
Average  weight  in  grams 

12 

Total 

ffr'g    Remarks 
wks. 

10 

0 

51 

54 

52 

47 

37 

see  (1)  below 

10 

6 

51 

61 

70 

70 

66 

61 

61 

62 

66 

15 

10 

8 

51 

65 

78 

84 

84 

83 

83 

86 

91 

40 

10 

10 

50 

65 

80 

90 

92 

91 

95 

100 

105 

55 

10 

12 

50 

67 

85 

97 

102 

105 

110 

115 

120 

70 

(1)  37  gms.=average  weight  at  death. 
56  days=average  age  at  death. 

It  will  be  noted  that  in  both  experiments  (Tables  V  and  VI) 
the  largest  difference  in  weight  gained  for  the  same  difference 
in  milk  fed  conies  between  6  cc.  and  8  cc.  In  order  to  prove 

13 


Conclusively  that  the  8  cc.  dose  is  the  best  one  to  use,  three 
lots  of  six  rats  each  were  given  diet  94  with  the  addition  of 
6  cc.,  7  cc.,  and  8  cc.  of  skimmed  milk  respectively  per  rat  per 
day.  The  animals  receiving  6  cc.  of  the  milk  gained  an  aver- 
age of  34  grams  in  eight  weeks,  those  receiving  7  cc.  averaged 
35  grams  gain  and  the  rats  fed  8  cc.  averaged  56  grams. 

Eight  cc.  of  skimmed  milk  per  rat  per  day  is  probably 
the  best  level  at  which  to  feed  in  order  to  detect  any  change 
in  the  vitamin  B  content  of  the  milk,  since  it  is  at  this  level 
that  we  get  the  largest  difference  in  the  total  gain  in  weight 
for  eight  weeks,  as  compared  with  the  next  lower  amount  fed. 
This  does  not  furnish  the  optimum  amount  of  vitamin  B,  since 
further  addition  of  milk  gave  increased  growth  in  many  cases 
and  in  the  general  average. 

In  another  experiment  the  amount  of  milk  given  was 
varied  in  accordance  with  the  weight  of  the  rat.  Each  animal 
received  0.05,  0.075,  0.10  or  0.125  grams  of  skimmed  milk 
powder  (0.50,  0.75,  1.00  or  1.25  cc.  reconstructed  milk)  per 
10  grams  of  rat  per  day.  The  amount  of  milk  fed  was  calcu- 
lated on  the  basis  of  the  weekly  weighings,  the  milk  fed  each 
day  for  a  week  being  determined  from  the  weight  of  the  rat 
at  the  beginning  of  the  week.  It  was  expected  that  this 
method  would  yield  more  consistent  results  and  that  the  varia- 
tion among  individuals  receiving  the  same  amount  of  vitamin 
B  would  be  lessened  by  this  method  of  feeding.  The  results, 
however,  do  not  bear  out  this  assumption.  A  comparison  of 
the  weight  curves  of  the  individual  rats  which  were  fed  the 
same  amount  of  milk  throughout  the  experimental  period  re- 
gardless of  body  weight,  with  those  of  the  rats  receiving  the 
graduated  doses,  shows  that  the  former  are  as  uniform  as 
those  in  the  other  series.  Table  VII  summarizes  the  results 
of  the  experiments  in  which  the  rats  received  the  graduated 
doses.  0.1  gram  of  milk  per  day  per  10  grams  of  rat  is  ap- 
parently the  best  level  at  which  to  feed  the  milk  by  this 
method,  since  for  the  same  difference  in  amount  of  milk  given 
a  greater  difference  in  weight  gained  is  observed  between  this 
and  the  next  lower  amount,  than  between  any  other  two  suc- 
cessive doses.  The  method  was  not  adopted  since  it  gave  no 

14 


assurance  of  having  any  advantage  over  the  former,  and  the 
former  was  easier  of  application. 

TABLE  VII. 
Diet  94  and  unheated  skimmed  milk  fed  in  fluid  state. 


N      Mi  Ik  per 

of  i°*r- 

of  rat  per 
day,  gms. 

4 

Age  in  weeks 
5      6      7      8      9      10    11 
Average  weight  in  grams 

Total 
12    ffring        Remarks 
wks. 

4 

0 

44 

50 

46 

36 

31 

see  (1)  below 

4 

0.05 

39 

50 

51 

50  ! 

46 

45 

47 

49 

59 

20 

4 

0.075 

44 

53 

58 

59 

60 

59 

66 

65 

73 

29 

4 

0.10 

43 

53 

63 

70 

74 

80 

89 

100 

110 

67 

4 

0.125 

43 

54 

61 

65 

71 

74 

83 

90 

106 

63 

(1)  31  gms.=average  weight  at  death. 
55  days=average  age  at  death. 

Feeding  the  Dried  Milk  Mixed  With  the  Rest  of  the  Diet. 

For  the  experiments  in  which  the  dried  milk  was  mixed  with 
the  rest  of  the  diet,  it  was  essential  to  determine  the  propor- 
tion in  which  the  milk  should  be  added  in  order  to  make  the 
test  for  possible  differences  in  vitamin  B  content,  most  deli- 
cate. The  starch  in  diet  94  was  replaced  by  skimmed  milk  in 
amounts  varying  from  2.5  per  cent,  to  30.0  per  cent.  The  diets 
are  given  in  Table  VIII. 

TABLE  VIII. 
Diet    94    98       97    96    95    99  100  101 

Casein    18     18  18     18     18     18     18     18 

Butter  fat  10     10  10     10     10     10     10     10 

Salt  mixture  (O.  &  M.)40  44  444444 

Skimmed  milk  powder..  0      2.5  5     10     15     20    25    30 

Starch    68    65.5  63     58     53    48    43     38 

The  rats  used  in  these  experiments  came  either  from 
mothers  on  Diet  13  or  13  M.  The  data  for  the  animals  from 
the  two  sources  have  been  summarized  separately  and  are 
given  in  Tables  IX  and  X  and  the  combined  results  in  Table 
XL  See  also  Chart  II,  Figure  I. 

15 


TABLE  IX. 
Rats  from  mothers  on  Diet  13. 


v 

Age  in  weeks 

Total 

JNj  O, 

of 

Diet 

Milk 
& 

4 

5 

6 

7  8 

9 

10 

11 

12 

ffring   Remarks 

rats 

/  " 

Average 

weight  in 

grams 

wks. 

6 

94 

0 

32 

36 

33 

32  29 

24 

see  (1)  below 

6 

98 

2.5 

41 

46 

46 

41  35 

30 

see  (2)  below 

6 

97 

5 

37 

42 

45 

44  42 

38 

see  (3)  below 

6 

96 

10 

40 

49 

57 

59  51 

46 

see  (4)  below 

5 

95 

15 

39 

52 

68 

74  71 

70 

74 

76 

see  (5)  below 

6 

99 

20 

36 

48 

64 

71  80 

86 

90 

93 

92 

56  K.  at  12  wks. 

6 

100 

25 

37 

47 

66 

78  94 

109 

118 

126 

132 

95   "  "  12  " 

6 

101 

30 

37 

53 

75 

94  114 

132 

143 

151 

159 

122   "  "  12  " 

K.  =  killed.  D.  =  died. 

(1)  24  gms.=average  weight  at  death. 
*69  days=average  age  at  death. 

(2)  30  gms.=average  weight  at  death. 
65  days=average  age  at  death. 

(3)  4  rats  D.  at  53,  63,  68,  69  days. 
2  rats  K.  at  12  weeks. 

(4)  2  rats  D.  at  61,  65  days. 
4  rats  K.  at  12  weeks. 

(5)  1  rat  D.  at  11  weeks. 
4  rats  K.  at  12  weeks. 

*  The  average  age  at  death  here  is  high,  due  to  the  fact  that  one  rat 
lived  100  days. 

TABLE    X. 
Rats  from  mothers  on  diet  13  M. 


No. 
of 
rats 

4 
6 
4 

4 
6 

5 
5 
5 

Age  in  weeks 
Diet    ^Ik4      5      6      78      9     10    11     12 

Average  weight  in  grams 
94        0     46    52    53    48    41     32 
98      2.5    51     57    60    59    52    47    36 
97       5     47    58    57    55    48    41    35 
96       10    48    61    70    69    62    54    47    40 
95       15    49    68    82    90    89    86    84    86 
99       20    51     71    89  101  103  109  107  103  102 
100       25    54    75    99  118  136  153  169  168  175 
101       30    56    73  103  125  146  165  182  197  204 

Total 

forl      Remarks 
wks. 
see  (1)  below 
see  (2)  below 
see  (3)  below 
see  (4)  below 
see  (5)  below 
51     K.  at  12  wks. 
121       "    "  12    " 
148      "   "  12    " 

K.  =  killed.                             D.  =  died. 
(1)  32  gms.=average  weight  at  death. 
62  days=average  age  at  death. 
(2)  36  gms.=average  weight  at  death. 
71  days=average  age  at  death. 
(3)  35  gms.=:average  weight  at  death. 
71  days=average  age  at  death. 
(4)  40  gms.=average  weight  at  death. 
75  days=:average  age  at  death. 
(5)  2  rats  D.  at  74  days. 
4  rats  K.  at  12  weeks. 

16 


TABLE  XL 

(Combined  results  of  Tables  IX  and  X.) 
Rats  from  mothers  on  Diet  13  and  13  M. 


No. 
of 
rats 

Diet 

Mi,k 

4 

Age  in  weeks 
56789 
Average  weight  in 

10    11 

grams 

Total 
12    «arin8       Remarks 

wks. 

10 
12 
10 
10 
11 
11 

94 

98 
97 
96 
95 
99 

0 
2.5 
5 
10 
15 
20 

38 
46 
41 
43 
44 
43 

42 
52 
48 
54 
61 
58 

41    38 
53    50 
50    AS 
62    63 

75    83 
75    85 

34    27 
44    33 
40    35 
55    50 
81    78 
91    96 

79 
98 

81 
97 

97      54 

see  (1) 
see  (2) 
see  (3) 
see  (4) 
see  (5) 
K.at 

below 
below 
below 
below 
below 
12  wks. 

11   100   25  45  59  81  96  113  129  141  145  151  106 


12 


11   101   30  46  62  88  108  129  147  161  172  179  133     "  12 

K.  =  killed.  D.  =  died. 

(1)  27  gms.=average  weight  at  death. 
66  days=average  age  at  death. 

(2)  33  gms.=average  weight  at  death. 
68  days=average  age  at  death. 

(3)  8  rats  D.  in  69,  68,  63,  53,  75,  73,  69,  67  days. 
2  rats  K.  at  12  weeks. 

(4)  6  rats  D.  at  65,  61,  75,  82,  72,  72  days. 
4  rats  K.  at  12  weeks. 

(5)  3  rats  D.  at  74,  74,  77  days. 
8  rats  K.  at  12  weeks. 

The  greatest  difference  in  rate  of  growth  for  a  difference 
of  5  per  cent,  in  the  amount  of  milk  contained  in  the  diet,  is 
found  between  20  and  25  per  cent.  Obviously  then  the  ration 
containing  25  per  cent,  of  skimmed  milk  powder  is  the  proper 
one  to  use  in  a  study  of  the  change  in  the  water  soluble  B 
content  of  skimmed  milk  powder  upon  heating. 

A  study  of  Tables  IX  and  X  further  shows  a  decided  dif- 
ference between  the  animals  from  the  two  sources.  Rats  from 
mothers  on  Diet  13  M  grew  faster  and  maintained  a  higher 
average  weight  than  the  animals  from  mothers  on  Diet  13 
alone.  The  young  rats  from  Diet  13  M  are  evidently  more 
vigorous  and  therefore  make  better  growth.  The  data  shows 
that  it  is  essential  either  to  use  rats  coming  from  mothers  on 
the  same  diet  or  to  take  the  same  number  of  rats  from  each 
diet  in  making  up  matched  lots  of  rats  for  comparison. 

A  typical  experiment  will  suffice  to  show  the  variation 
among  the  individual  animals  on  the  same  diet.  The  average 

17 


figures  for  8  cc.  milk  in  Table  VI  were  calculated  from  the 
data  given  in  Table  XII. 

TABLE   XII. 
Weights  of  10  rats  receiving  Diet  107  and  8  cc.  milk. 

Age  in  weeks  Total 

Rat  4        5        6        7        8        9       10      11       12     gain  for 

Weight  in  grams  8  weeks 


$5960.  . 

49 

63 

7Q 

86 

87 

80 

78 

81 

91 

42 

$5962.. 

31 

47 

63 

73 

78 

77 

74 

79 

83 

52 

55965 

55 

71 

81 

80 

82 

84 

8S 

89 

96 

41 

55968.. 

52 

67 

76 

80 

77 

73 

76 

82 

87 

35 

55994.. 

58 

73 

87 

93 

91 

84 

84 

87 

93 

35 

$5998.. 
56002.. 

50 
56 

56 
67 

68 
8? 

79 
89 

80 
94 

83 
96 

86 
Q3 

85 
92 

87 
89 

37 
33 

56040.. 

56 

70 

77 

82 

78 

7? 

77 

79 

85 

29 

56049.. 

56 

77 

88 

9? 

91 

97 

98 

103 

104 

48 

56055 

44 

63 

77 

8=i 

8S 

80 

83 

84 

92 

48 

av.  for  10  rats      51      65      78      84      84      83      83      86      91  40 

THE  EFFECT  OF  HEAT  UPON  VITAMIN  B  IN  MILK 
IN  THE  DRY  AND  FLUID  STATE. 

Experiments  with  Reconstructed  Skimmed  Milk  Fed  Sepa- 
rately from  the  Rest  of  the  Diet.  The  skimmed  milk  powder 
used  in  this  work  was  heated  both  dry  and  in  the  natural  fluid 
state,  in  the  manner  described  earlier  in  this  paper.  The  liquid 
milk  was  heated  for  six  hours  at  100°  C.  in  a  boiling  water  bath 
and  the  dry  milk  for  6,  24  and  48  hours  at  100°  C.  in  a  constant 
temperature  oven.  In  the  experiments  summarized  in  Table 
XIII  each  rat  received  8  cc.  of  the  reconstructed  milk  per  day  in 
addition  to  the  basal  ration  107,  8  cc.,  as  has  been  previously 
shown,  being  the  dose  which  would  be  most  likely  to  reveal 
any  modification  in  the  B  content  of  the  milk.  When  the  milk 
was  heated  dry  it  was  mixed  with  water  (1  gm.  milk  in  10  cc. 
water)  before  feeding.  The  advantage  of  this  method  of 
feeding  is  that  each  animal  receives  exactly  the  same  amount 
of  the  vitamin-containing  food  whereas  in  the  experiments  in 
which  the  milk  is  mixed  with  the  rest  of  the  food,  the  amount 
of  vitamin  the  rat  gets  depends  upon  how  much  food  it  eats. 

18 


TABLE  XIII. 
Diet  107  and  8  cc.  of  milk  treated  in  various  ways. 


No.  of 
rats 

10 
10 
10 
10 
10 

Milk 
treatment 

Unheated 
Heated  dry  6  hrs. 
Heated  dry  24  hrs. 
Heated  dry  48  hrs. 
Heated  in  fluid 
state  6  hrs. 

4 

53 
54 
54 

53 

54 

Ag 
5      6 
Average 
70    80 
69    80 
68    76 
67    75 

66    71 

je  in  weeks 
7      8      9      10     11 
weight  in  grams 

82    86    86    88    91 
83    87    88    89    91 
77    79    77    78    78 
78    82    85    85    86 

67    63    60    57    57 

12 

95 
96 
80 
90 

60 

Total 
gain  for 
8  weeks 

42 
42 
26 
37 

6 

The  results  in  Table  XIV  are  those  of  an  experiment  sim- 
ilar to  the  one  just  described,  but  in  place  of  8  cc.,  each  rat 
received  only  7  cc.  of  milk,  this  series  of  experiments  having 
been  completed  before  it  was  decided  that  8  cc.  would  be  the 
most  satisfactory  amount.  In  both  experiments  all  the  rats 
came  from  mothers  on  Diet  13  M. 

TABLE   XIV. 
Diet  94  and  7  cc.  of  milk  treated  in  various  ways. 


No. 
of 
rats 

Milk            Milk 
cc.           Treatment 

4 

Age  in  weeks                    Total 
5     6     7     8     9     10    11    12    gain  for 
Average  weight  in  grams       8  weeks 

3 

0 

42 

43 

44 

38 

32i 

3 

7 

Unheated 

45 

59 

70 

71 

71    68 

64 

64 

64 

19 

4 

7 

Heated  dry  6  hrs. 

45 

57 

72 

78 

74   71 

67 

64 

62 

17 

4 

7 

Heated  dry  24  hrs. 

46 

59 

75 

78 

74   71 

67 

63 

59 

13 

4 

7 

Heated  dry  48  hrs. 

40 

50 

61 

66 

69   68 

64 

57 

57 

17 

4 

7 

Heated  in  fluid 

state  6  hrs. 

40 

61 

68 

65 

59   53 

2 

1  32  grams  =  average  weight  at  death,  the  average  age  being  53  days. 

2  53  grams  =  average  weight  at  death,  the  average  age  being  62  days. 

There;  is  apparently  a  measurable  destruction  of  vitamin  B 
when  the  milk  is  heated  at  100°  for  6  hours  in  the  fluid  state. 
The  average  growth  curve  for  the  ten  rats  receiving  8  cc.  of 
the  reconstructed  milk  heated  six  hours  at  100°  C.  corresponds 
very  closely  to  the  average  curve  of  the  rats  receiving  6  cc.  of 
unheated  milk  (Tables  V  and  VI).  This  indicates  that  ap- 
proximately one-fourth  of  the  total  B  in  the  milk  was  destroy- 
ed when  the  fluid  milk  was  heated  for  six  hours  at  100°  C. 

19 


The  seasonal  differences  in  the  growth  of  the  rats  l>  2>  42  and 
the  possible  seasonal  variation  in  the  vitamin  content  of  the 
milk  1»  2>  41>  42>  43>  44  are  eliminated  here,  since  the  experiments 
(Tables  V  and  VI)  were  carried  on  at  the  same  time  in  the 
early  spring,  late  winter  milk  from  the  same  lot  being  used  in 
both  series.  The  four  rats  receiving  7  cc.  of  reconstructed 
milk  which  had  been  heated,  died  at  the  ages  of  50,  60,  66  and 
66  days  respectively,  while  the  rats  getting  the  same  amount 
of  unheated  milk  lived  till  the  end  of  the  twelfth  week  when 
they  were  killed.  This  is  additional  evidence  of  the  destruc- 
tion of  vitamin  B  by  this  heat  treatment. 

Dry  heat  applied  for  6,  24  and  48  hours  seems  to  have 
little  or  no  deleterious  effect,  as  the  weight  curves  of  the 
control  rats  receiving  unheated  milk  and  of  those  receiving 
the  heated  milk  are  approximately  the  same. 

Experiments  with  Milk  Mixed  with  the  Rest  of  the  Diet. 
Preliminary  experiments  reported  elsewhere  in  this  paper  in- 
dicate that  a  change  in  the  vitamin  B  content  of  skimmed  milk 
powder  may  be  most  easily  observed  if  the  milk  is  mixed  with 
the  rest  of  the  diet  in  the  proportion  equal  to  25  per  cent,  of 
the  entire  mixture.  Therefore  the  unheated  milk  of  Diet  100 
was  replaced  by  the  milk  heated  dry  for  various  periods,  6,  24 
and  48  hours.  The  animals  from  .mothers  on  Diet  13  M  and 
Diet  13  placed  on  these  diets  have  been  summarized  separate- 
ly in  Tables  XV  and  XVI  and  the  combined  results  are  given 
in  Table  XVII  and  Chart  II,  Figure  II.  It  will  be  noted  here 
again,  as  was  observed  from  the  results  of  the  experiments  in 
which  different  amounts  of  milk  replaced  the  starch  in  Diet 
94  (Tables  IX  and  X)  that  the  rate  of  growth  of  the  animals 
from  Diet  13  M  is  greater  than  that  of  the  animals  from 
Diet  13. 

The  total  food  consumed  by  the  animals  on  Diet  100  and 
the  diets  in  which  the  unheated  milk  of  this  ration  was  re- 
placed by  the  milk  heated  6,  24  and  48  hours  was  practically 
the  same  in  each  case.  The  rats  therefore  received  essentially 
the  same  number  of  calories  and  equal  amounts  of  protein,  of 
the  mineral  elements  and  of  the  vitamin  containing  food  on 
the  four  different  diets. 

20 


TABLE  XV.  . 
Rats  from  mothers  on  Diet  13. 


No. 

of 
rats 

Diet 

Milk                    Milk 
%                   Treatment 

4 

Age  in  weeks 
567        8       9       10       11 
Average  weight  in  grams 

Total 
12    gain  for 
8  weeks 

6 

94 

0 

43 

49   50 

47 

41    33* 

8 

100 

25 

Unheated 

39 

58   79 

90 

93    97 

99 

99 

98 

59 

8 

100 

25 

Heated  dry  6  hrs. 

39 

53   75 

87 

93  101 

103 

102 

106 

67 

8 

100 

25 

Heated  dry  24  hrs. 

39 

52   64 

81 

90  100 

105 

112 

118 

79 

8 

100 

25 

Heated  dry  48  hrs. 

39 

53   69 

83 

87    97 

100 

104 

108 

69 

*  33  gms.=average  weight  at  death,  the  average  age  at  death  being  60  days. 

TABLE  XVI. 
Rats  from  mothers  on  Diet  13  M. 


No. 
of 
rats 

Diet 

Milk 

% 

Milk 
Treatment 

4 

Age  in  weeks 
5       6       78       9        10      11 
Average  weight  in  grams 

12 

J 

Total 
gain  for 
3  weeks 

7 

94 

0 

52 

57    59 

54 

49 

42    35* 

7 

100 

25 

Unheated 

52 

71    92 

109 

129 

142  149 

164 

166 

114 

7 

100 

25 

Heated  dry  6  hrs. 

53 

73   92 

111 

122 

135  147 

162 

166 

113 

7 

100 

25 

Heated  dry  24  hrs. 

53 

70   88 

104 

116 

125  135 

141 

149 

95 

7 

100 

25 

Heated  dry  48  hrs. 

54 

67  83 

96 

111 

132  133 

145 

151 

97 

*  35  gms.=raverage  weight  at  death,  the  average  age  being  67  days. 

TABLE  XVII. 
(Results  of  Tables  XV  and  XVI  combined.) 


No. 
of 
rats 

Diet 

Milk 

% 

Milk 
Treatment 

4 

s 

Age  in  weeks 
67        8       9       10      11 
Average  weight  in  grams 

Total 
12     gain  for 
8  weeks 

13 

94 

0 

48 

53 

55 

51 

45    35* 

15 

100 

25 

Unheated 

45 

64 

85 

99 

110  118 

123  129 

130 

84 

15 

100 

25 

Heated  dry  6  hrs. 

45 

62 

83 

98 

106  117 

124  130 

134 

88 

15 

100 

25 

Heated  dry  24  hrs. 

46 

60 

77 

92 

102  112 

119  125 

132 

87 

15 

100 

25 

Heated  dry  48  hrs. 

46 

59 

76 

89 

98  113 

115  125 

128 

82 

*  35  gms.=average  weight  at  death,  64  days  being  the  average  age. 

The  conclusions  to  be  drawn  from  these  regarding  the 
heat  destruction  of  vitamin  B  in  the  dry  state,  confirm  those 
of  the  previous  experiment  in  which  the  heated  milk  was  fed 
separately.  The  average  gain  in  weight  for  eight  weeks  on 
a  control  diet  containing  unheated  milk  and  on  the  diets  con- 
taining the  milk  heated  for  6,  24  and  48  hours  is  the  same. 
There  is  evidently  no  destruction  of  the  vitamin  by  this  treat- 
ment or  the  amount  of  destruction  is  too  small  to  be  detected 
by  either  of  the  methods  employed  in  the  present  work. 

21 


SUMMARY 

A  quantitative  method  for  the  determination  of  relative 
amounts  of  vitamin  B  is  described.  The  work  comprises 
eleven  comparative  studies  with  a  total  of  thirty-eight  varia- 
tions of  the  basal  diet  and  includes  over  six  hundred  quanti- 
tative studies  of  the  growth  of  young  rats. 

An  adequate  basal  ration  is  described  which  contains 
optimum  amounts  of  all  the  food  factors  necessary  for  the 
growth  of  young  rats  with  the  exception  of  vitamin  B. 

The  most  advantageous  quantities  of  milk  to  feed  in  order 
to  detect  possible  differences  in  the  vitamin  B  content  were 
8  cc.  of  skimmed  milk  or  0.8  gram  of  skimmed  milk  powder 
per  rat  per  day  when  the  milk  was  fed  separately  from  the 
rest  of  the  diet ;  or  25  per  cent,  of  the  total  food- mixture  when 
the  skimmed  milk  powder  was  mixed  with  the  basal  ration. 

Feeding  the  basal  ration  under  the  conditions  described 
to  experimental  animals  of  suitable  age  and  size  and  suffi- 
ciently known  dietary  history,  it  is  believed  to  be  possible  to 
detect  a  variation  certainly  of  25  per  cent,  and  probably  of  15 
per  cent,  in  the  vitamin  B  content  of  the  food  tested. 

There  was  no  evidence  of  destruction  of  vitamin  B  in  milk 
powder  heated  dry  at  100°  C.  even  when  this  heating  was  con- 
tinued for  forty-eight  hours.  When  the  milk  was  heated  in 
the  fluid  state  for  six  hours  at  100°  C.  there  was  appreciable 
destruction  of  vitamin  B.  Apparently  about  one-fourth  of  the 
vitamin  was  destroyed.  Vitamin  B  is  therefore  very  stable 
to  heating  at  100°  C.  in  the  dry  state,  but  somewhat  less  stable 
when  heated  at  the  same  temperature  in  water  solution. 


22 


CHART  I. 


-30 


FIGURE  I. 


FIGURE  II. 
Figure  I. 
Average  gain  curves  of  rats  on  diet  94  plus  various  amounts 

of  skimmed  milk. 
Curve  1     Diet  94+15  cc.  milk 
'    94+12cc.     " 
1     94+lOcc.     " 
'    94+    8cc.     " 
'     94+   6cc.     " 
(     94+    4cc.     " 
'    94+   2cc.     " 


Figure  II. 
Average  gain  curves  of  rats  on  diet  107  plus  various  amounts 

of  skimmed  milk. 
Curve  1     Diet  107  +  12  cc.  milk 
'     107  +  10  cc.     " 
'     107+   8cc.     " 
'     107+   6cc.     " 
(     107  no  milk 


K.  =  killed. 


D.  =  died. 

The  last  point  on  the  curve  is  the  average  age  and  weight  at  death. 
Curves  6  and  7  in  Figure  I  could  not  be  completed  as  some  of  the  rats 
died  before  the  end  of  the  experimental  period  of  eight  weeks. 

23 


CHART  II. 


-30 


FIGURE  I. 


FIGURE  II. 


Figure  I. 


Curve  1     Average  gain  curve  of  rats  on  diet  101  (30%  milk) 


100  (25%  milk) 
99  (20%  milk 

95  (15%  milk) 

96  (10%  milk) 

97  (  5%   milk) 

98  (2.5%  milk) 
94  (no  milk) 


2 
"    3 


Figure  II. 

Curve  1  Average  gain  curve  of  rats  on  diet  100  (milk  unheated) 

"      "     "     "      "    100  (milk  heated 

6  hrs.  at  100°  C.) 
"      "     "     "      "    100  (milk  heated 

24  hrs.  at  100°  C.) 
"     4        "  "       "      "     "     "      "    100  (milk  heated 

48  hrs.  at  100°  C.) 
K.  =  killed.  D.  =  died. 

The  last  point  on  the  curve  is  the  average  age  and  weight  at  death. 
Curves  4,  5  and  6  in  Figure  I  could  not  be  completed  as  some  of  the  rats 
died  before  the  end  of  the  experimental  period  of  eight  weeks. 


24 


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25 


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practical  application.  Jour.  Biol.  Chem.  1920  XLIV 
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32  McDonald  and  McCollum :   The  cultivation  of  yeast  in  solu- 

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26 


33  McDonald  and  McCollum :    The  bios  of  Wildiers  and  the 

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34  Nelson,  Fulmer  and  Cessna :    The  nutritional  requirements 

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edge concerning  accessory  food  factors  (vitamines). 
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40  Osborne  and  Mendel :   Nutritive  value  of  the  wheat  kernel. 

Jour.  Biol.  Chem.  1919  XXXVII  572. 

41  Kennedy  and  Dutcher :  Vitamine  studies.  IX.  The  influence 

of  the  diet  of  the  cow  upon  the  quality  of  the  vitamines 
A  and  B  in  milk.  Jour.  Biol.  Chem.  1922  L  339. 

42  Hopkins :   Note  on  the  vitamine  content  of  milk.   Biochem. 

Jour.  1920  XIV  721. 

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vitamine.    Jour.  Biol.  Chem.  1918  XXXIV  537. 

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27 


VITA 

Adelaide  Spohn  was  born  in  Chicago,  Illinois,  May  25, 
1886.  In  1908  she  received  the  degree  of  B.  S.  from  the  Uni- 
versity of  Chicago  and  in  1913  the  degree  of  M.  S.  from  the 
same  University.  Since  1908  she  has  held  the  following  posi- 
tions :  Instructor,  Woodstock,  Illinois,  High  School  1908- 
1910;  Instructor,  Joliet,  Illinois,  Township  High  School  1910- 
1911 ;  Research  Assistant  to  Dr.  Oscar  Riddle  at  the  Univer- 
sity of  Chicago  and  Station  of  Experimental  Evolution  of  the 
Carnegie  Institution  of  Washington,  1911-1915;  Instructor  in 
Chemistry,  Teachers  College,  1915-1916;  Chemical  Assistant 
in  the  Department  of  Physiology,  Columbia  University,  1916 
to  February,  1918;  Instructor  in  Chemistry  Northwestern 
University,  February  1918-1919;  Instructor  in  Chemistry, 
Pratt  Institute,  1919-1920;  Research  Assistant  in  Food  Chem- 
istry, Columbia  University,  1920-1922. 

She  was  co-author  with  Dr.  Oscar  Riddle  of  the  following 
publications :  Studies  on  the  Physiology  of  Reproduction  in 
Birds.  II.  On  the  Chemical  Composition  of  White  and  Yel- 
low Egg  Yolk  of  the  Fowl  and  Pigeon,  Am.  Jour.  Physiol, 
1916,  XLI,  397 ;  Studies  on  the  Physiology  of  Reproduction  in 
Birds.  IV.  When  a  Gland  Functions  for  the  First  Time  is  its 
Secretion  the  Equivalent  of  Subsequent  Secretions  ?  Am.  Jour. 
Physiol.  1916,  XLI,  419. 

She  has  been  a  graduate  student  under  the  Faculty  of 
Pure  Science,  Columbia  University,  during  the  years  1915-16 
and  1920-22. 


28 


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